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Dendritic cell expanded t suppressor cells and methods of use thereofRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.), Eukaryotic CellDendritic cell expanded t suppressor cells and methods of use thereof description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070009497, Dendritic cell expanded t suppressor cells and methods of use thereof. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This Application claims the benefit of U.S. Provisional Application Ser. No. 60/551,354, filed Mar. 10, 2004, which is hereby incorporated in its entirety. FIELD OF THE INVENTION [0003] This invention relates to culture-expanded T suppressor cells and their use in modulating immune responses. This invention provides methods of producing culture-expanded T suppressor cells, which are antigen specific, and their use in modulating complex autoimmune diseases. BACKGROUND OF THE INVENTION [0004] Tolerance mechanisms for autoreactive T cells can be of "intrinsic" and "extrinsic" varieties. Intrinsic mechanisms include deletion and anergy of self-reactive T cells, while extrinsic mechanisms include different regulatory T cells that suppress other self-reactive T cells. One type of extrinsic suppressor is the CD25.sup.+ CD4.sup.+ T cell, which constitutes 5-10% of CD4.sup.+ peripheral T cells. These are produced in the thymus and maintain tolerance to self-antigens, as well as play a role in other immune responses, such as in infection, transplants and graft versus host disease. [0005] The transcription factor, FoxP3, is important for CD25.sup.+ CD4.sup.+ T cell suppressor activity, and children who are born with defective FoxP3 rapidly develop autoimmunity, such as, for example, autoimmune diabetes. Models for the study of autoimmunity have played a critical role in both the understanding of the pathogenesis, and the devising of therapeutic strategies for these diseases. In a mouse model of autoimmune diabetes, the non-obese diabetic (NOD) mice, for example, CD25.sup.+ CD4.sup.+ regulatory T cells inhibit diabetes development, making this extrinsic tolerance mechanism an attractive target to develop antigen-specific therapies for autoimmune disease. In an experimental model of multiple sclerosis mediated by transgenic T cells specific to myelin basic protein, CD25.sup.+ CD4.sup.+ T cells specific for this antigen showed better suppression of disease than CD25.sup.+ CD4.sup.+ T cells with TCRs specific for other antigens. These findings suggest a role for antigen-specific CD25.sup.+ CD4.sup.+T cells, in suppressing autoimmunity, though it remains unclear whether CD25.sup.+ CD4.sup.+ T cells of one antigen specificity, can suppress autoimmune disease, caused by T cell responses to many autoantigens. [0006] In vitro, CD25.sup.+ CD4.sup.+ T cells will suppress the proliferative or cytokine responses of naive CD25.sup.- CD4.sup.+ T cells, however, the CD25.sup.+ CD4.sup.+ T cells are themselves unable to proliferate, are anergized, when stimulated by antigen presenting cells (APCs), in vitro. It is therefore unclear how the numbers of regulatory T cells are sustained and expanded, in vivo. Further, CD25.sup.+ CD4.sup.+ T cell expansion in vitro is as yet limited, further confounding their application in therapeutic settings SUMMARY OF THE INVENTION [0007] This invention provides, in one embodiment, an isolated, culture-expanded T suppressor cell population, wherein the population expresses CD25 and CD4 on its cell surface. In one embodiment, the culture-expanded T suppressor cell population is antigen specific. In one embodiment, the culture-expanded T suppressor cell population expresses a monoclonal T cell receptor, or in another embodiment, expresses polyclonal T cell receptors. [0008] In one embodiment, this invention provides a method for producing an isolated, culture-expanded T suppressor cell population, comprising contacting CD25+ CD4+ T cells with dendritic cells and an antigenic peptide, an antigenic protein or a derivative thereof, or an agent that cross-links a T cell receptor on said T cells in a culture, for a period of time resulting in antigen-specific CD25+ CD4+ T cell expansion and isolating the expanded CD25+ CD4+ T cells thus obtained, thereby producing an isolated, culture-expanded T suppressor cell population. In one embodiment, the method further comprises the step of adding a cytokine to the dendritic cell, CD25+ CD4+ T cell culture, which in one embodiment, is interleukin-2. In one embodiment, the dendritic cells are selected for their capacity to expand antigen-specific CD25+CD4+ suppressor cells. [0009] According to this aspect of the invention, and in one embodiment, the dendritic cells are isolated from a subject suffering from an autoimmune disease or disorder, and in another embodiment, the antigenic peptide or antigenic protein is associated with the autoimmune disease or disorder. In one embodiment, the dendritic cells are isolated from a subject with an inappropriate or undesirable inflammatory response, and in another embodiment, the antigenic peptide or protein is associated with the inappropriate or undesirable inflammatory response. In one embodiment, the dendritic cells are isolated from a subject with an allergic response, and in another embodiment, the antigenic peptide or protein is associated with the allergic response. In one embodiment, the dendritic cells are isolated from a subject who is a recipient of a transplant, or in another embodiment, from a donor providing a transplant to said subject. In one embodiment, according to this aspect of the invention, the antigenic peptide or protein is associated with an immune response in the subject receiving a transplant from a donor. In one embodiment, the immune response is a result of graft versus host disease, or in another embodiment, the immune response is a result of host versus graft disease. [0010] In one embodiment, this invention provides a method for delaying onset, reducing incidence or suppressing an autoimmune response in a subject, comprising the steps of contacting in a culture CD25+ CD4+ T cells with dendritic cells and an antigenic peptide or an antigenic protein associated with an autoimmune response in a subject, for a period of time resulting in CD25+ CD4+ T cell expansion; and administering the expanded CD25+ CD4+ T cells thus obtained to a subject, wherein the isolated, expanded CD25+ CD4+ T cells suppress an autoimmune response in the subject, thereby delaying onset, reducing incidence or otherwise suppressing an autoimmune response. [0011] In one embodiment, this invention provides a method for downmodulating an immune response in a subject, comprising the steps of contacting in a culture CD25+ CD4+ T cells with dendritic cells and an antigenic peptide or an antigenic protein associated with an immune response in a subject, for a period of time resulting in CD25+ CD4+ T cell expansion; and administering the expanded CD25+ CD4+ T cells thus obtained to a subject, wherein said isolated, expanded CD25+ CD4+ T cells down modulate an immune response in said subject. In one embodiment one or more specificities, including a mixture of antigens derived from a (for diabetes) pancreatic beta cell line or islet tissue itself. [0012] In one embodiment, this invention provides a method for delaying onset, reducing incidence or suppressing an autoimmune response in a subject, comprising the steps of culturing an isolated dendritic cell population with an antigenic peptide or an antigenic protein associated with an autoimmune response in a subject and administering the dendritic cells to a subject, whereby the dendritic cells contact CD25+ CD4+ T cells, resulting in CD25+ CD4+ T cell expansion in the subject, wherein expanded CD25+ CD4+ T cells suppress an autoimmune response in the subject, thereby delaying onset, reducing incidence or suppressing an autoimmune response. In one embodiment one or more specificities, including a mixture of antigens derived from a (for diabetes) pancreatic beta cell line or islet tissue itself. [0013] In one embodiment, this invention provides a method for downmodulating an immune response in a subject, comprising the steps of culturing an isolated dendritic cell population with an antigenic peptide or an antigenic protein associated with an immune response in a subject and administering the dendritic cells to a subject, whereby the dendritic cells contact CD25+ CD4+ T cells, resulting in CD25+ CD4+0 T cell expansion in the subject, wherein expanded CD25+ CD4+ T cells downmodulate an immune response in the subject. [0014] In one embodiment, this invention provides a method for delaying onset, reducing incidence or suppressing an autoimmune response in a subject, comprising the step of contacting a dendritic cell population in vivo with an antigenic peptide or protein associated with an autoimmune response in the subject for a period of time whereby the dendritic cells contact CD25+ CD4+ T cells in the subject, stimulating antigen-specific expansion of the CD25+ CD4+ T cells in the subject, wherein expanded CD25+ CD4+ T cells suppress an autoimmune response in the subject, thereby delaying onset, reducing incidence or otherwise suppressing an autoimmune response. [0015] In another embodiment, this invention provides a method for modulating an immune response in a subject, comprising the steps of contacting a dendritic cell population in vivo with an antigenic peptide or protein associated with an immune response whose modulation is desired, whereby the dendritic cell population contacts CD25+ CD4+ T cells in the subject, wherein CD25+ CD4+ T cell contact promotes antigen persistence in said dendritic cell population in vivo, and the dendritic cell population with persistent antigen contacts effector T cells in the subject, wherein the effector T cells modulate an immune response associated with the antigenic protein or peptide thereby modulating an immune response in a subject. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 demonstrates DCs stimulate CD25+ CD4+ T cell growth. (A) CD25+ or CD25- CD4+ FACS-purified (top), DO11.10, OVA-specific T cells (1.times.104) were cultured 3d with spleen APCs (10.sup.5) or CD86+ mature DCs (5.times.10.sup.3) and anti-CD3 mAb, and 3H-thymidine uptake assessed (60-72 h). (B) As in (A), but T cells were from two OVA specific TCR transgenic mice, DO11.10 and OT-2, and the DCs were pulsed or not pulsed with 1 mg/ml OVA protein. (C) CD25+ CD4+ T cells from wild type BALB/C mice (closed diamonds) proliferate in response to DCs presenting anti-CD3 (right) but not OVA (left). (D, E) Day 6 marrow DCs were FACS separated into mature CD86high and immature CD86low CD11c+ subsets (D) and cultured with CD25+ CD4+ DO11.10 T cells (E) with OVA protein (1 mg/ml pulsed onto the DCs) or OVA 323-339 peptide (1 .mu.g/ml) continuously. One representative result of at least three experiments is shown. [0017] FIG. 2 demonstrates A large fraction of CD25+ CD4+ T cells are driven into multiple cell cycles by DCs. (A) As in FIG. 1, but the kinetics of proliferation (3H-thymidine and cell counts) were both followed. (B) CFSE labeled, T cells (1.times.104) were cultured 3d with 104 CD86+ mature BMDCs either OVA-pulsed (DC-OVA) or unpulsed (DC), prior to FACS analysis. (C) Quantitative estimation of the number of T cells entering cell cycle, and the number of mitotic events, was carried out as follows. CFSE-labeled CD25+ CD4+ T cells T cells (1.times.104) were cultured for 72 h with 1 mg/ml OVA pulsed CD86+ BMDCs (104), and analyzed for dilution of CFSE label (C). The percentage of total CD4+ events under each division peak (a) was experimentally determined (b). In this experiment, 24,000 live T cells were recovered, from which the absolute T cell count in each division peak at the time of harvest could be calculated (c). The absolute number of original, or precursor, T cells required to have generated these daughters is extrapolated by dividing the numbers of cells in column "c" by the number of divisions, 2n (d). The sum of the number of precursors giving rise to each peak represents the number of T cells at day 0 that entered cell cycle, which in this experiment was 3834 (the sum of column (d)) from a starting number of 10,000 T cells, giving a precursor frequency of 38%. The number of progeny in each peak (c) minus the number of precursors giving rise to the progeny (d) gives the number of mitotic events (e). The sum of these events represents the total number of cell divisions that occurred in the T cell subset by the time of harvest. (D) The experiment and calculation in (C) was carried out in a total of 6 experiments where the TCR stimulus was specific OVA antigen (n=3) or anti-CD3 antibody (n=3). [0018] FIG. 3 demonstrates the role of IL-2 in CD25+ CD4+ T cell proliferation. (A) 3H-thymidine uptake by CD25+ or CD25+ CD4+ T cells alone (top), or T cells stimulated by CD86+ DCs not pulsed (middle) or pulsed (lower) with OVA protein .+-. IL-2 or PC61 anti-IL-2R mAb. (B) As in (A) but IL-2 effects on 3H-thymidine uptake and cell counts were assessed with time. (C) As in (A), but anti-IL-2R mAb or control rat IgG was added to CD25+ CD4+ T cells stimulated with DCs from wild type (WT) or IL-2-/- mice plus OVA peptide at 1 .mu.g/ml for 3d. The numbers above the bars indicate the amount of IL-2 detected by ELISA in the same culture. (D) IL-2 production (ELISA) after stimulation with DC-OVA or DCs. Statistical significance was determined using the unpaired Student's t-test. *P<0.01. [0019] FIG. 4 demonstrates Membrane costimulation of CD25+ CD4+ T cells by DCs. (A) Comparison of T cell responses to live (top, T:DC ratio of 1:1) or formaldehyde fixed (bottom, T:DC=1:3) CD86+ mature marrow DCs plus DO11.10 peptide at 1 .mu.g/ml for 3d. Indicated concentration of anti-IL-2R Ab or control Ab were added to culture. Statistical significance was determined using the unpaired Student's t-test. *P<0.01. (B) Same as (A), but the activity of aldehyde-fixed DCs were studied with DCs that were charged with OVA (DC-OVA) or not (DC), and then added to CD25+ CD4+ and CD25- CD4+ T cells in the presence or absence of IL-2, with only the former subset responding to IL-2 in the absence of OVA (top left). (C) Marrow DCs (10.sup.4) were generated from wild type (WT) or CD80/CD86 knockout mice and matured in 50 ng/ml LPS prior to culture with CD25+ or CD25- CD4+ T cells (10.sup.4; purified from OT-II mice spleen and lymph node cells) for 3 days with or without 0.5 .mu.g/ml OVA peptide. The degree of proliferation was assessed by incorporation of 3H-thymidine for the last 12 h. One representative result of three independent experiments is shown. [0020] FIG. 5 demonstrates that CD25+ CD4+ T cells must contact DCs to proliferate actively. CFSE-labeled CD25+ CD4+ T cells (top) or CD25- CD4+ T cells (bottom) and the indicated stimuli were added to the inner and outer wells of transwell chambers, and the dilution of CFSE label per cell was followed by FACS after 3 days of culture. Dead cells were gated out by TOPRO-3 staining. One representative result of three independent experiments is shown. Continue reading about Dendritic cell expanded t suppressor cells and methods of use thereof... Full patent description for Dendritic cell expanded t suppressor cells and methods of use thereof Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Dendritic cell expanded t suppressor cells and methods of use thereof patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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